Metastasis is the major cause of breast cancer-related morbidity and mortality. The molecular mechanisms of metastasis, a complex and multi-step process, are yet to be fully elucidated, but epithelial-mesenchymal transition (EMT) is proposed to play a role in facilitating metastasis. Cells that undergo EMT have a greater capacity to migrate, to invade and to resist therapies. Zeb1, the most apical EMT transcription factor, is a powerful inducer of EMT via its transcriptional suppression of E-cadherin and of the microRNA-200 family expression. While the control of Zeb1 gene expression is currently being unravelled, the mechanisms of post-transcriptional Zeb1 regulation are not yet fully understood.
Siah, a family of E3 ubiquitin ligases, has recently been shown to have a role in tumourigenesis and metastasis. We found that Siah protein levels are reduced in breast cancer cells undergoing EMT. To assess whether Siah plays a functional role in the process of EMT, we generated and analysed murine Siah2-/- breast cancer cell lines. These cells display a mesenchymal phenotype in 2D and 3D cultures, despite unchanged levels in the gene expression of EMT transcription factors. Interestingly, Siah2-/- cells have increased Zeb1 protein abundance.
Mechanistically, we demonstrate that Zeb1 is a novel Siah-interacting protein, and that Zeb1 is polyubiquinated and targeted for proteasomal degradation by Siah. Human breast cancer cells undergo spontaneous EMT, associated with increased Zeb1 abundance, when Siah proteins are blocked by inhibitors or knocked down. This inverse correlation between Siah and Zeb1 proteins levels, as well as Siah expression and a mesenchymal gene signature, is also seen in breast and ovarian cancer patient samples.
Considering the importance of Zeb1 in controlling EMT, these findings of a mechanism of post-transcriptional regulation of Zeb1 by Siah ubiquitin ligases will greatly enhance our current understanding of EMT-inducing signalling networks.